Prieto et al.: Investigating cell type specific mechanisms contributing to acute oral toxicity Supplementary Data1 Tab. S1: Overview of collected information on specific target organ/system and general cytotoxicity for chemicals correctly assigned to the CLP acute oral toxicity category by the 3T3 NRU cytotoxicity assay General Nervous Cardiovascular GI Chemical Liver Lung Blood Kidney cytotoxicity system system system (±)-Propranolol hydrochloride x ax (4-ammonio-m-tolyl)ethyl(2- x x hydroxyethyl)ammonium sulphate 1,2,4-Trichlorobenzene x x 1,2-Dichlorobenzene x x 2,4,6- x x Tris(dimethylaminomethyl)phenol 2,4-Dichlorophenoxyacetic acid x x x 5,5-Diphenylhydantoin x x 5-Fluorouracil x x x x Acetophenone x Acetylsalicylic acid x x x x x x Acrolein x x Acrylamide x x x Ammonium chloride x ax ax Atropine sulfate monohydrate x x Benzaldehyde x x Cadmium (III) chloride x x x x x Caffeine x x x Chloroform x x ax x x x x Chloroquine bis(phosphate) x x Chlorpromazine x x x Codeine x x x x Colchicine x x x x Copper sulphate x x x Cupric sulfate pentahydrate x x Cyclosporin A x x x x Diazepam x Diphenhydramine hydrochloride x x Disopyramide x x Ethyl chloroacetate x x Ferrous sulphate x x x Glufosinate-ammonium x Glutethimide x ax x Hexachlorophene x x Lithium Carbonate x x x Lithium sulphate x x x Malathion x x Maleic acid x x Meprobamate x x Orphenadrine hydrochloride x x x x p-Benzoquinone x x x Phenol x x x x x Procainamide hydrochloride x x x Quinidine sulfate dehydrate x x x Resorcinol x x Rifampicin x x doi:10.14573/altex.1805181s2 ALTEX ##(#), SUPPLEMENTARY DATA 1 General Nervous Cardiovascular GI Chemical Liver Lung Blood Kidney cytotoxicity system system system Sodium Cyanate x x sodium oxalate x x x Sodium valproate x bx x x Thioridazine hydrochloride x x Valproic acid x x x x x GI: Gastrointestinal; CLP: Classification, labelling and packaging; NRU: Neutral Red Uptake; a Indirect effect: b chronic effect Tab. S2: Overview of collected information on specific target organ/system and general cytotoxicity for chemicals with CLP acute oral toxicity category under-predicted 3T3 NRU cytotoxicity assay General Nervous Cardiovascular GI Immune Chemical Liver Lung Blood Kidney cytotoxicity system system system system Potassium cyanide x x x (±)-Verapamil x hydrochloride 1-Phenyl-2-thiourea x x 1-Phenyl-3- x x pyrazolidone Barium chloride x x ax x Brucine x x x Chloral hydrate x x x x x cis- Diammineplatinum x x (II) dichloride D-Amphetamine x Dichlorvos x x x Disulfoton x Endosulfan x x Epinephrine x x hydrogen tartrate Fenpropathrin x Lindane x x x x Malononitrile x x ax x x x x x Mercury II chloride x bx x Methadone x x hydrochloride Nicotine x x Ochratoxin A x x x Paraquat dichloride x x x x Parathion ax x Pentachlorophenol x x x x Phenobarbital ax x ax physostigmine x Sodium pentobarbital ax x Sodium salt of x x x chloroacetic acid Strychnine x x Thallium sulphate x x x Theophylline x x x x Triethylenemelamine x x x Warfarin x x GI: Gastrointestinal; CLP: Classification, labelling and packaging; NRU: Neutral Red Uptake; a indirect effect; b corrosive ALTEX ##(#), SUPPLEMENTARY DATA 2 Tab. S3: Overview of collected information on specific target organ/system and general cytotoxicity for chemicals with CLP acute oral toxicity category over-predicted 3T3 NRU cytotoxicity assay Nervous Cardiovascular GI Immune Chemical Cytotoxicity Liver Lung Blood Kidney system system system system 17α- x x x Ethynyloestradiol 1-Naphthylamine x Amitriptilyne x x x hydrochloride Diquat Dibromide x x x x Haloperidol x x Maprotiline x x N-isopropyl-N'- phenyl-p- x phenylenediamine Tert-Butyl x x x hydroperoxide Triphenyltin x hydroxide GI: Gastrointestinal; CLP: Classification, labelling and packaging; NRU: Neutral Red Uptake Tab. S4: Specific target organ/systems and general cytotoxicity reported for the chemicals falsely predicted as negatives (i.e. LD50 > 2000 mg/kg) by the 3T3 NRU cytotoxicity assay General Nervous Cardiovascular GI Chemical Liver Lung Kidney cytotoxicity system system system Aconitine x x x Isoniazid x x x Diethylene glycol x x x Digoxin x Ethylene glycol x x x x Paraldehyde x x GI: Gastrointestinal; NRU: Neutral Red Uptake ALTEX ##(#), SUPPLEMENTARY DATA 3 Tab. S5: APOs outlined or fully developed with potential relevance to acute oral toxicity Adverse outcome MIE/Key Events Stressor References Cell death • Decompartmentalization Vinken and (Apoptosis/necrosis) • Direct mitochondrial inhibition Blaauboer, 1(AOP 205) • Narcosis 2017 In support of cytotoxicity as • Mitochondrial impairment (key event) general mechanism (non-cell specific) Haematotoxicity • Parent compound is converted to the 1(AOP 31) reactive metabolite and forms free radicals leading to oxidation of heme iron(II) in hemoglobin to iron(III) • Altered regulation of Alpha hemoglobin • Oxidative stress propagation • Damaging of Red blood cells; hemolysis • Formation of hemoglobin adducts • Down Regulation of Gulcose-6- phosphate dehydrogenase • Increase RBC congestion in liver • Increase Liver and splenic hemosiderosis • Decreased in hemoglobin, hematocrit, and red blood cell number (methaemoglobinemia) Acute liver response • PXR/SXR activation 1(AOP 11) • Key events unknown Cholestatic Liver Injury • Inhibition of Bile Salt Export Pump Vinken et al., 1(AOP 27) (ABCB11) 2013 • Activation of specific nuclear receptors, Transcriptional change • Bile accumulation • Cytokine Release • Increased Inflammation • Reactive oxygen species production • Peptide Oxidation Liver inflammation • Lysosome disruption • Iron compounds 1(AOP 144) • Peptide oxidation • ROS • Mitochondrial dysfunction 1 • o-methyl-serine • Cell injury/death dodecylamide • Cytokine Release hydrochloride (MSDH) • Inflammatory cells • alpha-tocopheryl succinate • 3-aminopropanal • artesunate • naphtharazine • Fluoroquinolones Decrease lung function • EGFR activation • ROS 1(AOP 148) • Trans differentiation of ciliated epithelial cells • Metaplasia of goblet cells • Goblet cell hyperplasia • Proliferation of goblet cells • SP1 Activation • Apoptosis of ciliated epithelial cells • Increased Mucin production • Chronic Mucus hypersecretion Increased mortality (Impaired • Impaired ion channels heart function) • Altered action potential 1(AOP 104) • Increased cardiac arrthymia Hypertension • Peptide Oxidation • ROS 1(AOP 149) • S-Glutathionylation, eNOS • Decrease in GTPCH-1 • Decrease in Tetrahydrobiopterin • Uncoupling of eNOS ALTEX ##(#), SUPPLEMENTARY DATA 4 Adverse outcome MIE/Key Events Stressor References • Depletion of Nitric Oxide • Impaired Vasodilation • Increase in Vascular Resistance • Decrease in AKT/eNOS activity Learning and impaired • Binding of agonist to ionotropic • Domoic acid memory glutamate receptors 4(AOP 48) • Mitochondrial dysfunction 1 • Cell injury/death • Neurodegeneration • Overactivation of NMDARs • Intracellular Calcium overload • Decreased in neuronal network function in adult brain • Neuroinflammation Epiletic seizures • Binding at picrotoxin site of iGABAR • Lindane 2(AOP 10) chloride channel • Endosulfan • Reduction in ionotropic GABA receptor • Picrotoxin chloride channel conductance • Dieldrin • Reduction of neuronal synaptic inhibition • Heptachlor • Generation of amplified excitatory • RDX postsynaptic potential (EPSP) • Fipronil • Occurrence of A paroxysmal depolarizing shift Parkinsonian motor deficits • Binding of inhibitor to NADH-ubiquinone • MPP+ 3(AOP 3) oxidoreductase (complex I) • Inhibition of NADH-ubiquinone oxidoreductase (complex I) • Mitochondrial dysfunction 1 • Impaired proteostasis • Neuroinflammation • Degeneration of dopaminergic neurons of the nigrostriatal pathway Acute mortality • Inhibition of acetylcholinesterase (AchE) • Organophosphates Russom et 1(AOP 16) • Accumulation of acetylcholine in • Carbamates al., 2014 synapses • Increased atrioventricular block and bradycardia • Increased respiratory distress/arrest • Induction of ataxia, paralysis, or hyperactivity Acute mortality • Modulation of sodium channel • Cypermethrin 1(AOP 96) • Prolonged depolarization of neuronal • Permethrin membrane • Esfenvalerate • Neuronotransmitter release • Tralomethrin • Muscle contraction • Bifenthrin • Increased ataxia, paralysis, or • Cyfluthrin hyperactivity • Lambda-Cyhalothrin Neurodegeneration • Binding to SH/seleno proteins • Mercuric chloride 1(AOP 17) • Oxidative Stress • Acrylamide • Glutamate dyshomeostasis • Acrolein • Cell injury/death • Methylmercuric(II) • Neuroinflammation chloride • Tissue resident cell activation • Thiomersal • Increased of pro-inflammatory mediators • Decrease of neuronal network function The acute neurotoxicity • Binding to the alpha subunit of voltage- • Pyrethroid Bal-Price et induced by binding of gated sodium channels. al., 2017 pyrethroid insecticides to • Changes in the kinetics of channel voltage-gated sodium opening and closing. channels • Alterations in excitability of neuronal membranes. • Dysregulation of neural networks (not in AOP-Wiki) Delayed neuropathy • Binding to neuropathy target esterase • Organophosphates Bal-Price et (NTE) al., 2015 Colony loss/failure • MIE unknown • Suppression of the immune system ALTEX ##(#), SUPPLEMENTARY DATA 5 Adverse outcome MIE/Key Events Stressor References 1(AOP 84) • Increased viral susceptibility • Impaired development Colony loss/failure • MIE unknown 1(AOP 85) • Suppression of the immune system • Increased viral susceptibility • Abnormal foraging activity and behavior Inhibition/activation of gastric • MIE unknown • Nonsteroidal anti- ulcer formation • Inhibition of